A site-specific labeling approach for systematic investigation of substrate binding and catalytic mechanisms employed by nucleic acid processing enzymes is outlined. Synthesis of nucleotide photoaffinity analogs-- 5-azidodeoxyuridine 1, 2- azidodeoxyadenosine 2, and 2-benzoyldeoxyadenosine 3 mono- (series A) and tri- (series B) phosphates and 5'-p-azidosulfonyl- benzoyldeoxyadenosine-- is investigated. Incorporation of photoaffinity analogs 1 and 2 into poly(dT)10 and poly(dA)10, respectively, by enzymic methodologies provides primer and exonuclease active site affinity labels. DNA polymerase I is employed as a test system to demonstrate the effectiveness of our approach in complimenting kinetic, crystallographic, mutagenesis, and FTNMR results in elucidating molecular contacts between polymerases and their substrates. Extensive molecular graphics and energy minimization techniques correlate affinity labeling results with a molecular description of nucleic acid processing-- DNA polymerization, processivity, and proofreading. These techniques will eventually allow elucidation of fundamental differences between eukaryotic, prokaryotic, and viral nucleic acid processing, and the design, based on first principles, of molecules of pharmacological interest which can specifically inhibit errant nucleic acid processing associated with human disease states.